Serotonin transporter (SERT) is responsible for lowering serotonin (5-HT) levels in the vicinity of synapses that release this neurotransmitter. In this process, 5-HT is transported into the cell from which it was released, where it is available for repackaging into synaptic vesicles. Drugs that inhibit SERT increase the level of synaptic 5-HT and lengthen the duration of its action. These drugs include abused substances such as cocaine, and therapeutic drugs such as antidepressants. Some drugs that are substrates for SERT, such as 3,4-methylenedioxymethamphetamine (a.k.a. "ecstasy") act through SERT to release 5-HT from neurons. From the profound behavioral consequences of these drugs, it is clear that regulation of SERT activity is likely to be a key event in normal brain physiology. Five years ago we found a mutant form of the transporter in patients with obsessive compulsive disorder. This mutant is apparently activated in a constitutive fashion by a pathway normally involving cyclic GMP (cGMP). We have since discovered that the defect represents a defect in the ability to remove a phosphate group at position 276 in the SERT molecule. This position is phosphorylated by a cGMP-dependent enzyme, PKG. As part of the long term goal to understand the structure and function of neurotransmitter transporters, this proposal outlines plans to investigate the association of enzymes that phosphorylate and dephosphorylate SERT, along with other components of the cGMP signaling pathway. The mechanism by which SERT conformation is affected by phosphorylation will also be investigated. All of these studies are directed to testing the hypothesis that production of cGMP activates a signaling pathway that phosphorylates and dephosphorylates SERT with the consequent increase and decrease in turnover rate. We will also test the prediction that conformational changes underlie the effects that 5-HT transport has on SERT interaction with regulatory proteins. PUBLIC HEALTH RELEVANCE: This proposal concerns a mechanism for regulation of serotonin transporter, a target for drugs of abuse and antidepressants. Defects in regulation were found in a mutant form of this transporter associated with psychiatric disorders. We aim to understand the structural and mechanistic events that are responsible for this regulation at a molecular level.